A new method for engineering bacterial chromosomes has emerged in recent years that takes advantage of the high proficiency of bacteriophage recombination systems acting on linear DNA substrates (for review, see Court et al., 2002). The λ Red recombination system, consisting of Bet (a ssDNA annealing protein) and Exo (a 5′-3′ dsDNA exonuclease) promotes gene replacement of electroporated linear DNA substrates into the Escherichia coli K-12 chromosome at a very high efficiency (Murphy, 1998; Murphy et al., 2000). Inactivation of host RecBCD exonuclease activity, either by mutation or production of the anti-RecBCD λ Gam function, is required for efficient Red-promoted recombination with linear dsDNA substrates (Murphy, 1998). Zhang and co-workers (Zhang et al., 1998), using the E. coli rac prophage RecET recombination system, recognized that PCR-generated substrates with as little as 40 bp of homology could serve as efficient substrates for gene replacement in E. coli. The use of such substrates has also been demonstrated with the λ Red system, with Red and Gam being supplied from either a prophage (Yu et al., 2000), a low-copy number plasmid (Datsenko and Wanner, 2000), or from a ΔrecBCD::Ptae-gam-red chromosomal substitution (Murphy, 1998; unpublished observations). The high efficiency of Red and RecET-promoted recombination with such short regions of homology has allowed E. coli geneticists to perform oligo-directed gene replacements that yeast geneticists have performed for years (Baudin et al., 1993; Lorenz et al., 1995; Wach et al., 1994).